Osteogenesis-Related Behavior of MC3T3-E1 Cells on Substrates with Tunable Stiffness.

Osteogenic differentiation of cells has considerable clinical significance in bone defect treatment, and cell behavior is linked to extracellular matrix stiffness. This study aimed to determine how matrix stiffness affects cell morphology and subsequently regulates the osteogenic phenotype of osteogenesis precursor cells. Four PDMS substrates were prepared with stiffness corresponding to the elastic modulus ranging from 0.6 MPa to 2.7 MPa by altering the Sylgard 527 and Sylgard 184 concentrations. MC3T3-E1 cells were cultured on the matrices. Cell morphology, vinculin expression, and key osteogenic markers, Col I, OCN, OPN, and calcium nodule, were examined. The activity and expression level of Yes-associated protein (YAP) were evaluated. Results showed that cell spreading exhibited no correlation with the stiffness of matrix designed in this paper, but substratum stiffness did modulate MC3T3-E1 osteogenic differentiation. Col I, OPN, and OCN proteins were significantly increased in cells cultured on soft matrices compared with stiff matrices. Additionally, cells cultured on the 1:3 ratio matrices had more nodules than those on other matrices. Accordingly, cells on substrates with low stiffness showed enhanced expression of the osteogenic markers. Meanwhile, YAP expression was downregulated on soft substrates although the subcellular location was not affected. Our results provide evidence that matrix stiffness (elastic modulus ranging from 0.6 MPa to 2.7 MPa) affects the osteogenic differentiation of MC3T3-E1, but it is not that "the stiffer, the better" as showed in some of the previous studies. The optimal substrate stiffness may exist to promote osteoblast differentiation. Cell differentiation triggered by the changes in substrate stiffness may be independent of the YAP signal. This study has important implications for biomaterial design and stem cell-based tissue engineering.